We are submitting this application in response to an RFA 93-HL-15-H. The long term objective of this application is to develop rabbit models of cardiovascular disease using the combined approach of (i) developing a transgenic animal model by microinjection of transgene constructs, and (ii) developing rabbit embryonic stem cells for the production of loss- of-function animal model by homologous recombination in the future. This application takes advantage of the close collaboration between two investigators with different backgrounds and expertise. The P.I. (L. Chan) has been interested in the application of molecular biology to atherosclerosis research and has had considerable experience in generating transgenic animals. The Co-P.I. (A. Bradley) is a molecular embryologist and geneticist who has been a pioneer in ES cell and gene- targeting technology. We have worked closely together for the past year- the P.I. helping the Co-P.I. in his pilot experiments on the establishment of rabbit ES cells, and the Co-P.I. helping the P.I. in an unrelated project on gene-targeting in mice. We will continue our collaboration in this application on the development of rabbit models of cardiovascular disease by genetic manipulation. The major objectives of this application are: (1) To optimize techniques for production of transgenic rabbits by microinjection of exogenous DNA. (2) To produce transgenic rabbits that overexpress human 15-lipoxygenase and study their susceptibility to atherosclerosis development. (3) To establish embryonic stem (ES) cells from rabbits, and finally (4) to initiate experiments on knocking out the rabbit apoE gene. There is good evidence that oxidative modification of low density lipoprotein (LDL) increases its atherogenicity. Lipoxygenases (LO) are involved in the oxidative process. 15-LO overexpression in fibroblasts in vitro has been shown to be associated with an enhanced capacity to oxidize LDL. We will produce transgenic rabbits with the human 15-LO cDNA driven by a macrophage-specific promoter (lysozyme cis-acting elements). These animals will be characterized with respect to their susceptibility to diet-induced atherosclerosis. In parallel with our transgenic rabbit generation, we will develop embryonic stem (ES) cells from rabbits. The Co-P.I. has extensive experience in developing ES cells from mice. The definition of a rabbit ES cell line will be: "a permanent tissue culture cell line capable of multiple population doublings (100) that exhibits an undifferentiated embryonic phenotype in culture yet is capable of contributing to both the soma and the germ line of a chimaeric rabbit". Once we have established a rabbit ES cell line, we will initiate experiments on apolipoprotein E knockout in these cells. The overall long term goal of this proposal is to generate rabbit models for atherosclerosis research.